Brief Summary
This video provides a step-by-step guide on performing a total leukocyte count (TLC) experiment, also known as white blood cell count. It covers the necessary apparatus, the procedure for collecting and diluting the blood sample, charging the Neubauer chamber, counting cells under a microscope, and performing the calculations to determine the final WBC count. Additionally, the video addresses common questions related to WBC diluting fluid composition, definitions of leukocytosis, leukopenia, and leukemia, causes of physiological leukocytosis, and potential sources of error in the experiment.
- Explains the step-by-step procedure for performing a total leukocyte count experiment.
- Covers the apparatus, sample preparation, Neubauer chamber charging, and cell counting.
- Discusses calculations, definitions of related conditions, and potential sources of error.
The video begins with an introduction by a first-year MBBS student who will demonstrate the total leukocyte count experiment. The required apparatus includes spirit, cotton, a lancet, Turk's fluid (WBC diluting fluid), a Neubauer chamber, and a white blood cell pipette. The Neubauer chamber being used is the improved type.
The first step is to sterilize the ring finger to prevent infection. The ring finger is preferred because it is not directly connected to the bursa. A lancet is used to prick the finger, and the blood is collected using the WBC pipette up to the 0.5 mark. If excess blood enters the pipette, it can be removed by tapping the pipette against the palm. The hand and pipette tip are then cleaned with cotton.
Turk's fluid is added to the pipette up to the 11 mark to dilute the blood sample. The pipette is then held horizontally, and the fluid is mixed thoroughly by moving the arms to ensure proper dilution. A drop of the mixture is discarded to remove excess diluting fluid before charging the Neubauer chamber.
A drop of the diluted blood is placed on the Neubauer chamber, allowing it to spread across the chamber via capillary action. The slide is then left to settle for one to two minutes to allow the cells to distribute evenly.
The Neubauer chamber is placed on the microscope, and a 10x lens is used to count the white blood cells. The WBCs are counted in the four corner squares of the chamber. The dimensions of each square are 1 mm in length and breadth, and 0.1 mm in height, resulting in a volume of 0.1 mm³. The video shows a diagram of a completed Neubauer chamber with the counted WBCs in each square.
The video explains how a dilution factor of 20 is achieved. Starting with 0.5 ml of blood and adding diluting fluid up to the 11 ml mark results in a 1:20 dilution. This means that 1 ml of blood is diluted into 20 ml of fluid.
The volume of each Neubauer chamber square is 1 mm x 1 mm x 0.1 mm = 0.1 mm³. For the four squares counted, the total volume is 0.4 mm³. If the total number of WBCs counted in these four squares is 198, then the number of WBCs per mm³ is calculated. The calculation involves dividing the number of WBCs (198) by the volume (0.4 mm³) and then multiplying by the dilution factor (20). The final WBC count is approximately 9900 per mm³.
The WBC diluting fluid, also known as Turk's fluid, contains glacial acetic acid, methyl violet, and distilled water. Glacial acetic acid destroys the membranes of RBCs, WBCs, and platelets. Methyl violet stains the nuclei of the white blood cells, and distilled water acts as a solvent.
Leukocytosis is an increase in the WBC count, leukopenia is a decrease in the WBC count, and leukemia is an excessive number of white blood cells in the peripheral blood system. The normal range of WBCs is 4,000 to 11,000. In leukocytosis, the count exceeds 11,000; in leukopenia, it falls below 4,000; and in leukemia, it is well above 50,000.
Physiological causes of leukocytosis include pregnancy, menstruation, muscular exercise, diurnal variation, and puberty. Pathological causes include acute and chronic infections, allergic reactions, and leukemia. Pyogenic bacterial infections are the most common cause of leukocytosis. Pathological causes of leukopenia include typhoid fever, sulfa drugs, and irradiation.
WBCs are produced in the bone marrow. The bead in the pipette aids in mixing the blood with the diluent, helps identify the pipette (white bead for WBCs), and indicates whether the pipette is dry or wet. In a dry pipette, the bead moves freely, while in a wet pipette, it sticks to the surface.
Possible sources of error include using a wet pipette, sucking blood beyond the 0.5 mark, formation of air bubbles, and sedimentation of RBCs. To minimize errors, ensure the pipette is clean and dry, tap the pipette against the palm to adjust the blood level, handle the blood carefully to avoid air bubbles, and roll the pipette in the palms to mix the blood and fluid well. The first few drops of the mixture are discarded because they mostly contain the diluting fluid.
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Transcript
Full transcript of “Determination of WBC count (TLC -Total leucocyte count) | MUHS | #mbbs #practical #hematology”. Click a timestamp to open the video at that moment. Need another video? Use the YouTube transcript generator.
0:00 my name is anish kulkarni i'm a first year mbbs student from hbtmc and cooper hospital today i'll be performing uh the total leukocyte count experiment so first we'll check the apparatus for the experiment and then we'll i'll tell you the procedure for the experience so the basic things we require are spirit some cotton a lancet other than that we require turks fluid or the white blood cell diluting fluid the nubus chamber and the white blood cell pipette this is the improved nua so the first thing we need to do is sterilize your hand to make sure that you do not get any infections
0:45 be sure to take the ring finger for for any pricking procedure as it is not connected to the bursa i use the pipette and this uh suck to take the blood inside the pipet up till the 0.5 mark
1:34 no if any excess blood enters the pivot then use the palm of your hand and just tap the pipette till the blood is out of the pipette then clean off your hand and the tip of the pipette using a cotton then add the turks fluid up till the 11 mark which you can see here up to here
2:22 and then uh use the pipette and suck the diluting fluid up till the 11 mark which you can see here okay now further dial hitting fluid until the eleventh point then pull with your arms like this as it contains mostly the diluting fluid
3:07 then uh take a drop here and then drop it in the new pass chamber it will slide across the whole numbers chamber due to the capillary action now let the slide settle for one to two minutes after you are done charging the new boss chamber and you are done waiting for one to two minutes uh place the nubus chamber in on the microscope as like this then to count use a 10x microscope lens to count place this at 10.
3:53 [Music] so this is the diagram for the nubus chamber so here here here and here we count the white blood cells so start from here uh and uh when you see in the microscope whatever you see however many white blood cells you see start writing it here and then complete this whole chamber dimension so the dimensions of the nubus chamber are 1 mm of length and bread and 0.1 mm of height so the total volume comes to 0.1 mm per so this is what the completed nuvar chamber will uh diagram will look like with each square having some number of white blood cells which you see in the microscope now i'll tell how the dialogue factor of 20 is achieved so first you take a 0.5
4:40 ml worth of blood and then you add 11 ml worth of diluting factor then you discard some amount of the fluid in the pipette in fluid so if you multiply it by 2 then 1 ml of the blood contains 20 ml of the diluting fluid that is why 20 are dilating factors so now coming to the calculation part so the volume of each universe chamber is 1 into 1 into 0.1 0.1 mm is the height 1 mm 1 mm is the length and 1 mm is the breadth which comes down to 0.1 mm cube now this is for one box so for four boxes it is going to be into four which is equal to 0.4 mm cube
5:26 of volume now after calculating the total number of wbcs from these boxes it comes down to 198 so if 0.4 mm cube has 198 wbcs so 1 mm cube let's take this as x so x is going to come out as 198 divided by 0.4 now this this part won't give us the final answer because we'll have to multiply this by the diluting factor as well which is which is explained before and hence after calculating this the total number of rbcs will come down as 9900 per mm cube
6:16 so the first question is what is the composition of wbc diluting fluid and what are the functions of each constrictor so the wbc dilating fluid is also known as turks fluid it contains glacial acetic acid which destroys the membranes of our bc wbc and platelets the second thing it contains its methyl violet which colors the fluid and also stains the nuclei of the white blood cells and third is distilled water which is used as a solvent coming to the second question in his definition of leukocytosis leukopenia and leukemia so the leukocytosis is increase in a wbc number leukopenia is decreasing wbc number and leukemia is excess amount of white blood cells in the peripheral blood system okay the normal range of white blood cells is four thousand to eleven
7:03 thousand uh so in leukocytosis uh the range uh the number increases above eleven thousand that is why it is increasing white blood cells number that is leukocyte second is leukopenia in it uh the white blessing decreases below four thousand and third is leukemia in which the white blood cell count increases well above fifty thousand question three is enumerate the causes of physiological leukocytosis uh the physiological causes are one pregnancy or menstruation for women second is muscular exercise third is diurnal variation and the fourth is puberty pathological causes of leukocytosis pathological causes of leukocytes are acute and chronic infection the allergic reactions and leukemia
7:49 the pyogenic bacterial infection is the most common cause of increase in white blood cells that is leukocytosis pathological causes of leukopenia typhoid fever sulphur drugs and irradiation and then where are the wbcs produced in the body the wbcs are produced in the bone marrow what is the function of this bead in the bulb the first function is it aids in mixing of the blood with diluent the secondly it helps in identifying the pipette as you can see the blade is white in color so it is for the white blood cells and third it tells whether the pipette is dry or not in a dry pet the bead will uh go around freely but in a wet pipette it will stick to the surface and will not
8:35 uh move around freely so now we'll discuss the possible sources of error and how to minimize them one of the sources of error is if the pipet is wet clean and dry the pipette okay and second source of error is if blood is sucked beyond the 0.5 mark and tap it against your palm to go below the 0.5 mark and if there is formation of air bubbles while sucking off blood well pick the hand carefully and then suck the bell and then if if there is sedimentation of rbc's in the bulb roll the pipette in your palms to mix the blood and fluid well and why do we need to discard the first few drops of rbc because they mostly contain the dilating fluid
9:20 so that's it for the white blood count experiment thank you for listening to us
